PB1763: THE CELLULAR UPTAKE OF CPX-351 BY SCAVENGER RECEPTOR CLASS B TYPE 1-MEDIATED NONENDOCYTIC PATHWAY IN LEUKEMIA CELLS.
Authors: Hiroaki Araie, Naoko Hosono, Takahiro Yamauchi
Journal: HemaSphere (2023)

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: The proper uptake of drugs in liposome formulations into target cells markedly impacts therapeutic efficacy. The protein corona (PC), formed by the adsorption of serum proteins onto the liposomal surface, binds to specific surface receptors of target cells, influencing the uptake pathway. The mechanism of uptake of CPX-351, a liposomal agent of cytarabine and daunorubicin, in leukemia cells remains poorly understood. Aims: This study aimed to analyze the PC of CPX-351 in vitro and explore the mechanisms of the uptake pathway in leukemia cell lines. Methods: The uptake of CPX-351 was assessed using a range of leukemia cell lines (HL-60, K562, and THP-1), drug-resistant sublines (HL-60/AD expressing multidrug resistance protein and K562/ADM expressing P-glycoprotein), and patient samples. The PC of CPX-351 mixed with fetal bovine serum was analyzed using nanoLC-MS/MS. The uptake of CPX-351 was measured by flow cytometry using daunorubicin fluorescence. The uptake pathway of CPX-351 was inhibited by chlorpromazine (CPZ), an inhibitor of clathrin-mediated endocytosis (CME), and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), an inhibitor of macropinocytosis and that blocks lipid transport-1 (BLT-1), a selective inhibitor of scavenger receptor class B type 1 (SR-BI). SR-BI is a nonendocytic pathway that takes up only liposome content. In addition to inhibiting uptake via SR-BI, BLT-1 has the unique characteristic of increasing the SR-BI binding affinity for liposomes in a dose-dependent manner. Results: The major components of the PC of CPX-351 were apolipoprotein A-II (relative proportion: 47.34%, ± standard error (SE):6.7) and A-I (5.25% ± 0.52), which bind to SR-BI. SR-BI was expressed in HL-60 (26.3 arbitrary units (AU) ± SE: 3.8), K562 (37.2 AU ± 3.4), and THP-1 (141.3 AU ± 24.7). The uptake of CPX-351 was observed in HL-60 (10.1 AU ± 0.2), K562 (8.7 AU ± 0.2), and THP-1 (13.9 AU ± 0.6). CPZ treatment significantly reduced the uptake of CPX-351 in HL-60, K562, and THP-1 cells compared with that in control cells, with reductions of 18.5%, 50.6%, and 34.6%, respectively (p<0.01). Similarly, EIPA treatment significantly reduced uptake by 9.9%, 43.4%, and 30.9%, respectively (p<0.01). Additionally, treatment with low-dose BLT-1 (400 nM) significantly reduced the uptake by 17.0%, 27.7%, and 24.4%, respectively (p<0.01). Conversely, the addition of high-dose BLT-1 (40 µM) significantly enhanced the uptake of HL-60, K562, and THP-1 cells by more than two-fold compared with control cells, with increases by 334.6%, 249.3%, and 266.8%, respectively (p<0.01). Enhanced uptake was significantly inhibited by the addition of EIPA, with reductions of 30.4%, 33.4%, and 29.3%, respectively (p<0.01). In each cell line, CPX-351 alone tended to decrease uptake when combined with CPZ rather than with EIPA. However, when CPX-351 was combined with high-dose BLT-1, the addition of EIPA decreased the uptake compared with CPZ. In HL-60/AD and K562/ADM cells, while the uptake of CPX-351 was lower than that in the parental cells, the addition of high-dose BLT-1 significantly increased the uptake by more than two-fold compared with CPX-351 alone. In patient samples, CPX-351 uptake was associated with SR-BI expression. The addition of high-dose BLT-1 also increased uptake, but not in samples with low SR-BI expression. Summary/Conclusion: The primary pathways for the uptake of CPX-351 were CME, macropinocytosis, and the SR-BI-mediated nonendocytic pathway. Our findings indicate that the enhanced binding between SR-BI and CPX-351 activated different pathways, such as macropinocytosis, distinct from CPX-351 alone. SR-BI may serve as a biomarker for CPX-351 therapy, and its combination with high-dose BLT-1 may be a useful finding to augment therapeutic efficacy. Keywords: Chemotherapy, Acute myeloid leukemia, Endocytosis, Nanoparticle